CN110835614B - Bifidobacterium lactis GKK2, composition containing same and application of composition in improving allergic asthma - Google Patents

Bifidobacterium lactis GKK2, composition containing same and application of composition in improving allergic asthma Download PDF

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CN110835614B
CN110835614B CN201811083035.0A CN201811083035A CN110835614B CN 110835614 B CN110835614 B CN 110835614B CN 201811083035 A CN201811083035 A CN 201811083035A CN 110835614 B CN110835614 B CN 110835614B
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bifidobacterium lactis
gkk2
composition
allergic asthma
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CN110835614A (en
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陈劲初
陈炎炼
林诗伟
陈彦博
王启宪
侯毓欣
石仰慈
林静雯
陈雅君
江佳琳
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Grape King Bio Ltd
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Abstract

The invention provides bifidobacterium lactis GKK2, a composition containing the bifidobacterium lactis GKK and application of the bifidobacterium lactis GKK to improving allergic asthma. The bifidobacterium lactis of the invention has acid-, bile-and/or heat-resistant capacity, and colony formation per milliliter is singly located in a culture medium between pH3.2 and pH2.0 and is about 5x10 8 cfu/ml about 5X10 in liquid medium supplemented with 0.3% bile salts 9 cfu/ml and/or heating at a specific temperature for a time of between greater than 0 minutes and about 15 minutes in a liquid medium of about 5x10 7 cfu/ml to 5x10 9 cfu/ml. The composition containing the strain can be used for reducing respiratory resistance, reducing the content of nonspecific antibody IgE in serum, reducing the content of specific antibody IgE in serum, slowing down the contraction degree of trachea and improving the content of glutathione reductase (GSH reductase) in liver tissue fluid so as to improve allergic asthma.

Description

Bifidobacterium lactis GKK2, composition containing same and application of composition in improving allergic asthma
Technical Field
The invention relates to a lactobacillus for improving allergic asthma, a composition containing the same and application thereof; more specifically, it relates to a method for preparing a bacterium from Lactobacillus bifidusBifidobacterium lactis) A composition comprising the species is produced and administered to a subject for reducing respiratory resistance, reducing non-specific antibody IgE content in serum, reducing specific antibody IgE content in serum, slowing the degree of tracheal contraction and increasing glutathione reductase content in liver tissue fluid to ameliorate allergic asthma.
Background
Allergic reaction
Allergic reactions are those in which the immune system is overreactive to substances that are otherwise harmless and cause acute or chronic inflammation of the tissues, and serious or even organ dysfunction. Common allergic diseases such as allergic asthma, allergic rhinitis, urticaria, atopic dermatitis …, etc.
Allergic asthma
Allergic asthma is a respiratory disease, the prevalence of asthma is continuously increasing with the rapid deterioration of the global environment, and the main symptoms are dyspnea, cough, chest distress, wheezing and the serious cases may be fatal. When the Allergen in the environment enters the human body through various ways, the Allergen first undergoes a Sensitization process, that is, when the Allergen is contacted with the Allergen (Allergen) for the first time, the Allergen is combined with specific antigen cells, when the specific antigen cells present the Allergen to T lymphocytes, the T lymphocytes differentiate into Th2 cells, and then many inflammation-related cytokines are released, and at the same time, the B lymphocytes are stimulated to produce antibody IgE (immunoglobulin E), which is attached to the surface of mast cells (map cells) along with blood, so that the antibody IgE has allergic constitution. When the human body is exposed again to the same allergen, the allergen binds to IgE on the surface of the mast cells, and stimulates the mast cells to release a plurality of inflammation-related mediators, such as histamine, leukotriene (LT), interleukin (IL), and the like, which directly or indirectly cause inflammatory reaction of the respiratory tract. When inflammatory reaction occurs, the airway tube is swollen and the smooth muscle around the airway is contracted, so that the airway tube is narrowed, and along with the increase of mucous gland secretion, once mucous is filled in the air gap inside the airway tube, the airway tube is contracted suddenly, and asthma is caused.
Glutathione
Glutathione (GSH) is a tripeptide containing gamma-amide linkages and sulfhydryl groups, and is composed of glutamic acid, cysteine, and glycine. This substance is present in almost every cell of the body and has the function of maintaining the body's normal immune system. The glutathione reductase can reduce the oxidized state (GSSG) of glutathione into the reduced state (GSH) of glutathione, thereby increasing the concentration of GSH in vivo and achieving the effect of strengthening the immune system.
Bifidobacterium genus
Bifidobacterium genus ]Bifidobacterium) Is widely used in the digestive tract, vagina, oral cavity, etc. of human and animals. It is gram positiveThe cells are not moving, are rod-shaped, have a bifurcated end at times, and are strictly anaerobic. The bacteria were isolated from the faeces of healthy infants as early as 1899, and subsequent studies have found that a part of specific strains of the bacteria can be used as probiotics for applications in the fields of food, medicine and feed.
Previous studies have revealed that a few lactic acid bacteria promote INF-gamma expression, thereby inhibiting the expression levels of IL-4, IL-5 and specific IgE associated with allergy, and reducing Th2 immune responses, thereby alleviating allergy symptoms. It has also been pointed out that continuous use of lactic acid bacteria for four months can increase IFN-gamma secretion in people with congenital allergic constitution to improve allergy. Clinical trial results in recent years have even found that early intake of probiotics can reduce the occurrence or symptomatic relief of atopic dermatitis in infants and relatively reduce the occurrence of allergic asthma. In addition, the former scholars useL. acidophilusDouble-blind tests are carried out, and the allergic asthma symptoms caused by dust mites and pollen can be effectively reduced. However, none of the above mentioned strains can ameliorate the problem of excessive constriction of the allergic airways.
Disclosure of Invention
The invention provides a bifidobacterium lactisBifidobacterium lactis) The bacterium has acid, bile salt and/or heat resistance, and colony formation per ml is about 5×10 in culture medium having pH 3.2-pH 2.0 8 cfu/ml about 5X10 in liquid medium supplemented with 0.3% bile salts 9 cfu/ml and/or heating at a specific temperature for a time of between greater than 0 minutes and about 15 minutes in a liquid medium of about 5x10 7 cfu/ml to 5x10 9 cfu/ml。
Preferably, the strain is bifidobacterium lactisBifidobacterium lactis) GKK2 and is preserved in China general microbiological culture Collection center (CGMCC) (address: the institute of microbiology, national academy of sciences, china) of North Chen West Lu 1, beijing, chaoyang, date of preservation: classification naming of 2018, 01, 12: bifidobacterium lactis @Bifidobacterium lactis) (accession number BCRC910826 was deposited at the institute of food industry, proprietary).
The invention provides a composition for effectively improving allergic asthma, which comprises bifidobacterium lactisBifidobacterium lactis) GKK2 the strain is preserved in China general microbiological culture Collection center (BCRC 910826, collection number is preserved in the institute of food industry, national institute of advanced as French of financial institutions) with a collection number of CGMCC No. 15205.
The invention also provides a composition for improving allergic asthma, which comprises bifidobacterium lactisBifidobacterium lactis) GKK2 the active substance, wherein the bifidobacterium lactis GKK2 is deposited with the China general microbiological culture Collection center (accession number BCRC910826, deposited with the institute of food industry, proprietary, with accession number CGMCC No. 15205).
Preferably, the active material is prepared by the following method:
(a) Inoculating lactobacillus colony (colony) into a culture medium for solid state culture to form lactobacillus thallus; a kind of electronic device with high-pressure air-conditioning system
(b) Inoculating the lactobacillus cells cultured in the step (a) into a liquid culture medium to perform liquid culture.
Preferably, the method further comprises the steps of:
(c) Centrifuging the liquid medium containing lactobacillus cells cultured in step (b) to obtain bacterial sludge; a kind of electronic device with high-pressure air-conditioning system
(d) And (3) freeze-drying the bacterial sludge obtained in the step (c).
Preferably, the temperature of step (b) is from 35 to 50 ℃, the aeration rate is from 0 to 1vvm nitrogen or carbon dioxide, the rotation speed is from 10 to 100rpm and/or the number of days of cultivation is from 16 to 24 hours.
Preferably, the pre-freeze temperature of the freeze drying of step (d) is from-196 to-40 ℃.
Preferably, the above composition comprises an additive selected from the group consisting of: excipients, preservatives, diluents, fillers, absorption promoters, sweeteners or combinations thereof.
Preferably, a composition comprising an active substance of bifidobacterium lactis GKK2 or GKK is a pharmaceutical product, feed, beverage, nutritional supplement, dairy product, food product or health food product.
Preferably, the composition is in the form of a powder, lozenge, granulate, suppository, microcapsule, ampoule, liquid spray or plug.
Experiments prove that the invention proves that the bifidobacterium lactis isBifidobacterium lactis) The strain GKK of (2) has a use for improving allergic asthma.
Preferably, the allergic asthma improvement for the above use is a decrease in respiratory resistance in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
Preferably, the allergic asthma ameliorated by the above use is a decrease in the serum level of non-specific antibody IgE in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
Preferably, the allergic asthma improvement for the above use is a decrease in the IgE content of specific antibodies in serum of a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
Preferably, the allergic asthma improvement for the above use is a reduction in the degree of tracheal constriction in the subject to whom bifidobacterium lactis GKK2 is administered, relative to the subject to whom bifidobacterium lactis GKK2 is not administered.
Preferably, the allergic asthma improvement for the above use is an increase in glutathione reductase (GSH reductase) content in liver tissue fluid of a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
Drawings
FIG. 1 shows the colony count of the acid resistance test results.
FIG. 2 shows colony numbers of the bile salt tolerance test.
FIG. 3 shows the colony numbers of the heat-resistant test results.
Fig. 4 shows the experimental schedule.
Fig. 5 shows the airway resistance test results.
Figure 6 shows the non-specific antibody IgE content in serum.
Figure 7 shows the specific antibody IgE content in serum.
Fig. 8 shows a section of tracheal tissue.
FIG. 9 shows the content of glutathione reductase (GSH reductase).
Microbial preservation for patent procedures:
bifidobacterium lactis GKK2 of the present invention:
preservation date: 2018, 01, 12;
preservation unit: china general microbiological culture Collection center (CGMCC);
deposit unit address: the institute of microbiology, national academy of sciences, north chen xi lu 1, 3, the region of the morning sun in beijing;
preservation number: CGMCC No. 15205;
classification naming: bifidobacterium lactis @Bifidobacterium lactis）。
Detailed Description
Bacterial origin
The lactic acid bacteria used in this experiment were bifidobacteria belonging to the genus bifidobacteriumBifidobacterium lactis) Also known as bifidobacterium lactis. In a preferred embodiment, the strain isolates intestinal specimens from three month old healthy male infants who are breast-fed, and the male infants are not treated with drugs or probiotic-related products that may affect gastrointestinal function and the bacterial phase of the specimens from birth to sampling. The sample has normal appearance and is in yellow brown pasty state, and accords with the characteristic of breast milk. After the sample is produced, the sample is placed into an anaerobic tank related device containing an anaerobic gas production bag so as to maintain the activity of anaerobic bacteria in the sample, and the sample is transported to a laboratory under a refrigerating environment for strain separation and screening operation. In a preferred embodiment, the strain is preserved in China general microbiological culture collection center (CGMCC) (address: north Xielu No. 1, 3, national academy of sciences of China) with a preservation number of CGMCC No. 15205 at 1/12 of 2018: classification naming of 2018, 01, 12: bifidobacterium lactis @Bifidobacterium lactis) (1.12.2018 with accession number BCRC910826 to the institute of food industry, proprietary).
Screening culture
The anaerobic jar containing the specimen was opened in an anaerobic operation table in an anaerobic environment, and about 2 g of the sample was sampled with a sterilized medicine spoon, and the sample was put into 200 ml of sterile physiological saline solution to homogenize the sample. Diluting the homogenized sample to 10 4 ~10 7 Multiplying power, then, the sample dilution was taken out and the dilution was subjected to pouring culture in TOS-MUP medium. The TOS-MUP culture medium is utilized to screen and separate the Phanerochaete specific bacteria in the sample, so that the Enterobacter interference in the sample is effectively eliminated, the defect of the traditional BIM-25 culture medium is overcome, and the Phanerochaete specific bacteria screening rate is 100%.
Placing the plate cultured by the pouring culture method into an anaerobic jar, opening an anaerobic gas bag for oxygen consumption to seal the anaerobic jar, then removing an anaerobic operation table, and placing the plate in a constant temperature incubator at 37+/-0.5 ℃ for culturing for 44-48 hours.
After culturing, the anaerobic jar is moved to an anaerobic operation table for unsealing and selecting and storing single strains, more than 100 single strain colonies are selected from each sample individually for separation and culture for storing and in vitro test cell evaluation, and peripheral blood mononuclear cells, spleen primary cells and mast cells are subjected to in vitro evaluation and CISD2 longevity gene activation test, and screening tests are carried out by a plurality of in vitro tests to screen high-functionality lactic acid strains with the functions of improving immunity and relieving in vivo anaphylactic reaction and skin anaphylactic reaction. The novel probiotic bacterial strain with multiple physiological effects improved is obtained by separation, and can be used for fermenting milk or vegetable juice and preparing probiotic powder for direct administration so as to improve a plurality of abnormal physiological reactions.
Phenotypic analysis
Study of the differences in phenotype between lactic acid bacteria and other species compared GKK by acid, bile salt and heat resistance tests.
Acid resistance test
GKK2 BCRC 17394 purchased from the research center of biological resource conservation and research institute of food industry, and Bifidobacterium animalis subspecies lactis Bi 04%B. animalis subsp. LactisBi 04, deposited in the American type strain preservation center under the accession number ATCC SD 5219), bifidobacterium animalis subspecies BB-12%B.animalis subsp. LactisBB-12 deposited in the German center for preservation of microorganism strains under the number DSM 15954) and Bifidobacterium animalis subspecies Bi07%B.animalis subsp. LactisBi07, deposited as ATCC SD 5220 at the American type culture Collection) for a total of 5 strains. By adding HCl to MRS liquid medium, the pH value of the original MRS liquid medium with pH value of about 6.5 is adjusted to be 3 different pH values: about pH3.2, pH 2.4 and pH 2.0. The strains were inoculated in these media of different pH values and after 3 hours of incubation at 37℃the colony formation numbers were counted.
As a result, as shown in FIG. 1, the number of GKK and other 4 strains could reach 5X10 in the original pH culture (about pH 6.5) 9 cfu/ml. At pH3.2, the bacterial count of all strains was slightly reduced, and GKK2 showed no significant difference from the other 4 strains. When the pH value is reduced to 2.4 and 2.0, the numbers of BCRC 17394, BB-12 and Bi07 are reduced to about 6 to 7 times, which are significantly lower than GKK (P) with the number of bacteria maintained above 8 times<0.05). Accordingly, the number of viable bacteria of GKK2 is significantly greater than that of other strains in an acidic environment, and the experimental results show that GKK has better acid resistance than other strains, so that GKK has better gastric acid resistance when passing through the stomach.
Test for bile salt resistance
GKK2 BCRC 17394 purchased from the research center of biological resource conservation and research institute of food industry, and Bifidobacterium animalis subspecies lactis Bi 04%B. animalis subsp. LactisBi 04, deposited in the American type strain preservation center under the accession number ATCC SD 5219), bifidobacterium animalis subspecies BB-12%B.animalis subsp. LactisBB-12 deposited in the German center for preservation of microorganism strains under the number DSM 15954) and Bifidobacterium animalis subspecies Bi07%B.animalis subsp. LactisBi07, deposited as ATCC SD 5220 at the American type culture Collection) for a total of 5 strains. Inoculating the strains into MRS liquid culture medium containing 0.3% bile salt, soaking at 37deg.C for half an hour, observing and counting the number of strains。
As shown in FIG. 2, the numbers of GKK2 and other 4 strains can reach 5×10 in the original MRS liquid medium 9 cfu/ml. In MRS with 0.3% bile salt, the numbers of BCRC 17394, bi 04 and Bi07 were all significantly lower than GKK (P)<0.05 The bacterial count of BB-12 was not statistically significantly different from that of GKK2. Accordingly, the number of viable bacteria of GKK2 is significantly greater than that of other strains in a bile salt environment, and the experimental result shows that GKK has better bile salt resistance than other strains, so that GKK has better bile salt resistance when passing through the digestive tract in vivo.
Heat resistance test
GKK2 BCRC 17394 purchased from the research center of biological resource conservation and research institute of food industry, and Bifidobacterium animalis subspecies lactis Bi 04%B. animalis subsp. LactisBi 04, deposited in the American type strain preservation center under the accession number ATCC SD 5219), bifidobacterium animalis subspecies BB-12%B.animalis subsp. LactisBB-12 deposited in the German center for preservation of microorganism strains under the number DSM 15954) and Bifidobacterium animalis subspecies Bi07%B.animalis subsp. LactisBi07, deposited as ATCC SD 5220 at the American type culture Collection) for a total of 5 strains. These strains were inoculated into MRS liquid medium, heated at 70℃for 5, 10, 15 minutes, and the number of strains was observed and counted.
As a result, as shown in FIG. 3, the numbers of GKK2 and other 4 strains could reach 5X10 in the unheated condition (0 min) 9 cfu/ml. After heating at 70℃for 5 minutes, the numbers of BCRC 17394, bi 04 and Bi07 were all significantly lower than GKK 2.sup.2 (P)<0.05 The bacterial count of BB-12 was not statistically significantly different from that of GKK2. After heating at 70℃for 15 minutes, the numbers of other 4 strains were significantly lower than GKK (P)<0.05). Accordingly, the number of viable bacteria of GKK2 is significantly more than that of other strains in a high-temperature environment, and the experimental result shows that GKK2 has better heat resistance. Moreover, lactic acid bacteria are generally not heat resistant and must remain active at low temperatures, and this test shows that GKK even has the properties of being heat resistant and long-lasting at normal temperatures, which is very helpful for applying GKK2 to process development.
Strain culture
The bifidobacterium lactis is inoculated with colony (colony) on a solid medium to activate the strain. In a preferred embodiment, the solid medium is MRS agar. After the growth of the thalli is completed, the fresh thalli and the solid culture medium are inoculated into a conical flask containing the liquid culture medium for liquid culture. In a preferred embodiment, the culture is carried out in liquid form at a temperature of 35 to 50℃and at a aeration rate of 0 to 1vvm of nitrogen or carbon dioxide at a rate of 10 to 100 rpm. In a preferred embodiment, the time for liquid culture is 16 to 24 hours, more preferably 18 hours. In a preferred embodiment, the liquid medium is MRS liquid medium. In a preferred embodiment, the liquid medium is formulated as shown in Table 1 below.
TABLE 1
Preparation of freeze-dried powder
After the strain of the lactobacillus grows in the liquid culture, collecting a liquid culture medium containing lactobacillus thallus, and centrifuging to obtain bacterial mud. In a preferred embodiment, the liquid medium containing the lactic acid bacteria is centrifuged at a rate of 1000 to 15000 rpm. Mixing the obtained bacterial mud with protective agent (skimmed milk powder with 6-30% of protective agent), lyophilizing, and storing at low temperature. In a preferred embodiment, the pre-freeze temperature of the freeze-drying is set at-196 to-40 ℃. In a preferred embodiment, the lyophilization time is from 16 to 72 hours. In a preferred embodiment, the storage temperature is from-30℃to 0 ℃. The stored lactobacillus freeze-dried powder is taken as a raw material for the following cell experiments, namely one of the aspects of the lactobacillus active substances claimed in the scheme. The aspect of the lactic acid bacteria active material claimed in the present application also includes the state of the culture solution containing the bacterial cells obtained by subjecting the bacterial cells to liquid culture.
Experimental animals and design
The experimental animals purchased 24 animals in total from 5-week-old male rats of Balb/c variety. The independent IVC (individual ventilated caging system) mouse raising system with mice raised in special mouse rooms is characterized in that the room temperature of an animal room is maintained at 22+/-2 ℃, the relative humidity is maintained at 40-60%, the lighting period is controlled by an automatic timer to be a dark period (dark period) from 07:00 to 19:00, and the lighting period (light period) from 19:00 to 07:00 is controlled by the automatic timer. The feed and the sterile reverse osmosis water are taken by mice freely. The mice were kept for 2 weeks to adapt to the environment before the experiment started.
Animal allergy patterns can be established in animal experiments by sensitization (sensitization). In the experiment, allergen is taken as antigen, and aluminum hydroxide (Aluminum hydroxide) is taken as adjuvant, and the allergen is injected into sensitized animals by subcutaneous injection. To establish the establishment of an animal allergy pattern, blood was collected before and after one week of antigen injection, and serum was taken from the blood sample to analyze the antigen-specific antibody concentration. If necessary, the serum should be sensitized three to four times to make the concentration of antigen-specific antibody reach the standard for establishing allergic mode, so that the subsequent experiment detection can be carried out. The animal pattern of respiratory tract allergies is established by spray sensitization, whereby the animal inhales the allergen and contacts the allergen from the respiratory tract.
Ovalbumin antigen induced allergy and asthma in mice by mixing ovalbumin antigen (OVA, 500 μg/ml) with incomplete adjuvant (Incomplete Freund's adjuvant) at 1:1, uniformly mixing and emulsifying the components in equal amount, and filtering the components by a sterile filter membrane for immediate use. Each group of mice was subcutaneously injected with this allergen on days 30, 40 and 50 of the experiment, and 10 minutes of respiratory inhalation (inhalation) was performed on days 45 and 55 with 2% OVA allergen spray to induce allergic asthma in the mice.
24 male Balb/c mice at the age of 5 weeks in the experiment are divided into a control group fed with physiological saline, a sensitized group induced by Ovalbumin (OVA), and an experiment group of high and low dose groups fed with bifidobacterium lactis GKK after induced by Ovalbumin (OVA), wherein the total number of the groups is 4, and 6 of the groups are respectively. Feeding and taking the mice by bifidobacterium lactis GKK, and tube irrigation, wherein the calculation of the feeding dose is based on the equivalent dose conversion coefficient of the surface area ratio of the experimental animal to the human body, and the conversion coefficient of the mice to the human is 12.3. The mice were weighed once a week, and after each group calculated the average body weight, daily doses were taken for each 20 mg and 100 mg (about 100 and 300 hundred million viable bacteria per person per day) per sample adult (calculated as 60 kg body weight) supplied by the manufacturer, and the daily tube-infused mice were converted to the following formula:
daily product dose (g) = { adult dose/60 kg (adult body weight) } ×mouse body weight (kg) ×12.3 (conversion factor of mouse to human)
The doses of bifidobacterium lactis GKK2 fed to the experimental group calculated by the above formula are shown in table 2 below:
TABLE 2
The experimental samples for mice fed were prepared by adding the above dose of bifidobacterium lactis GKK2 to 0.9% physiological saline. The stainless steel feeding needle is soaked in 75% alcohol solution at ordinary times, and is cleaned and wetted with 0.9% physiological saline before use. The mice of each group were respectively fed with 0.2 ml volumes of physiological saline (control group) or different concentrations of bifidobacterium lactis GKK2 solution by tube-feeding with a stainless steel feeding needle sleeved on a 1 ml sterile plastic syringe. Each group of mice was daily tube fed the above samples for a total of 60 days of continuous administration during the execution period. The detailed experimental schedule is shown in figure 4.
Mice were sacrificed on day 62 to obtain blood and serum was isolated and preserved. After collecting macrophages in the abdominal cavity of the mice, the abdominal skin was cut off and blood samples were collected from the portal vein using a 1 ml syringe. The whole blood sample of the mouse is collected and placed in a test tube containing an anticoagulant, and the white blood cells in the blood are subjected to differential counting. In addition, the whole blood sample of the mouse was collected in a test tube containing no anticoagulant, left standing at room temperature for 1 hour, and centrifuged at 1000 rpm for 10 minutes to obtain a supernatant sample, namely serum. Serum is stored at-80 ℃ for a long time, and specific antibodies induced by immunoglobulin and allergen in the serum are analyzed later.
Respiratory resistance test
Mice were subjected to noninvasive measurements of changes in respiratory resistance of mice on the day prior to sacrifice using the Whole lung function test system (white-body plethysmograghy, WBP) available from Buxco corporation. The resistance change is based on the value measured during the respiration of the mouse, and is analyzed by a computer to obtain an enhanced pause (Penh) serving as a lung function index, and if the value is higher, the resistance of the respiratory tract is larger. First, the mice were placed in an animal cabin, and the basal Penh value of the mice was measured for three minutes. Next, after exposing the mice to nebulized 0.9% NaCl for three minutes, penh values were measured for three minutes. Again, after three minutes exposure of the mice to the nebulized nonspecific bronchoconstriction stimulator acetylmethylcholine (6.25 mg/ml), the Penh value (PenhMch) was measured for three minutes and the above procedure was repeated with increasing concentrations of acetylmethylcholine (12.5 mg/ml, 25 mg/ml, 50 mg/ml). The experimental results are expressed in terms of Penh Ratio in the present experiment, and the data analysis is calculated by dividing the PenhMch readings obtained for each concentration of acetylmethylcholine by the penhmic readings obtained for saline.
Analysis of immunoglobulin IgE content in serum
The immunoglobulin IgE antibody content in the serum was quantified by a sandwich enzyme-linked immunosorbent assay (sandwich-ELISA). Firstly, preparing various anti-mice monoclonal antibodies with a proper amount by using a coating solution (pH 9.6), covering the anti-mice monoclonal antibodies on a 96-well micro-analysis plate (Nunc-immune plate), standing for 1 hour, washing the unbound monoclonal antibodies by using a washing solution (Wash buffer), and filling the part unbound by the monoclonal antibodies by using a blocking solution (blocking buffer) of 200 mu l/well. After 30 minutes of reaction at room temperature, the reaction mixture was rinsed with a washing solution (wash buffer) and 100. Mu.l of mouse serum or immunoglobulin IgE standard of known concentration was added. After the reaction of the serum or immunoglobulin IgE standard of the mice for 1 hour at room temperature, the mice are washed by a washing liquid (wash buffer), and then 100 μl/well of a secondary antibody of anti-immunoglobulin IgE which is connected with HRP at a proper concentration is added. After 30 minutes of reaction at room temperature, washing with a washing solution (wash buffer), followed by reaction with 100. Mu.l of TMB substrate, 15 timesAfter a minute of coloration, 100 μl of 2N H was added 2 SO 4 The color reaction was terminated and the absorbance was measured at a wavelength of 450 nm. According to the standard curve displayed by the immunoglobulin IgE standard substance and the light absorption value, the concentration of the antibody in serum can be estimated by an interpolation method through a formula obtained after regression operation.
Analysis of allergen-induced specific antibody IgE content in serum
The allergen-induced specific IgE content in the serum was quantified by means of a sandwich enzyme-linked immunosorbent assay (sandwich-ELISA). Firstly, a proper amount of anti-mice monoclonal antibody is prepared by using a coating solution (pH 9.6), 100 mug/mL Ovalbumin (OVA) antigen is added, the mixture is covered on a 96-well micro-analysis plate (Nunc-immune plate), after standing for 1 hour, the unbound monoclonal antibody is washed by using a washing solution (wash buffer), and then the part not bound by the monoclonal antibody is filled by using a blocking solution (blocking buffer) of 200 mug/well. After 30 minutes of reaction at room temperature, the reaction mixture was rinsed with a washing solution (wash buffer) and 100. Mu.l of mouse serum or immunoglobulin IgE standard of known concentration was added. After the reaction of the serum or immunoglobulin IgE standard of the mice for 1 hour at room temperature, the mice are washed by a washing liquid (wash buffer), and then 100 μl/well of a secondary antibody of anti-immunoglobulin IgE which is connected with HRP at a proper concentration is added. After reaction at room temperature for 30 minutes, washing with a washing solution (wash buffer), followed by reaction with 100. Mu.l TMB substrate, after 15 minutes for coloration, 100. Mu.l 2N H was added 2 SO 4 The color reaction was terminated and the absorbance was measured at a wavelength of 450 nm. According to the standard curve displayed by the immunoglobulin IgE standard substance and the light absorption value, the concentration of the allergen-induced specific antibody in the serum can be estimated by interpolation according to the formula obtained by regression operation.
Tissue section
After embedding the tracheal tissue in paraffin and tissue sections, hematoxylin and eosin staining was performed (hematoxylin and eosin stain, H & E stain). The morphology was observed under a microscope.
Analysis of glutathione reductase (GSH reductase) content
The livers of mice were quantitatively placed in HEPES buffer cell culture medium, and samples were kept at-80℃until the analysis of glutathione reductase (GSH reductase) was awaited, and the content thereof was analyzed by a commercially available ELISA method.
Biometric analysis
The experimental results reported in this test were expressed as mean.+ -. SD and were statistically analyzed in a computer using SPSS 12.0 software suite, the differences between the groups were determined using Duncan's multiple range test, the criterion for significant differences was p <0.05, and the experimental results were plotted using SigmaPlot 10.0 software.
Experimental results
For the respiratory resistance test, the experimental results of each group are shown in table 3 and the statistics are shown in fig. 5:
TABLE 3 Table 3
From the above results, when the concentration of the atomized bronchoconstriction stimulator, acetyl methylcholine, was increased, the respiratory resistance of the sensitized group, which was intraperitoneally injected and inhaled with OVA, was greatly increased, and significantly higher than that of the control group, indicating that the sensitized group was successfully established in OVA-sensitized mouse mode. The experimental group to which GKK2 was administered was sensitized mice, and the respiratory resistance rise was low as the concentration of the atomized bronchoconstriction stimulator acetylmethylcholine was increased. The instructions showed that GKK administration reduced airway pressure caused by OVA allergen, which was an improvement in the level of compression of asthma. In addition, the increase in respiratory resistance was less pronounced when the concentration of GKK2 administered was increased, indicating that GKK has a concentration-dependent effect on the relief of allergic asthma.
The experimental results for the non-specific antibody IgE content in serum are shown in table 4 and the statistics are shown in fig. 6:
TABLE 4 Table 4
The non-specific antibody IgE antibodies in the serum of the sensitized mice (sensitized group) were significantly increased compared to the control group. The non-specific antibody IgE content in serum of the sensitized mice (high and low dose experimental groups) administered GKK by tube feeding was significantly reduced (p < 0.05) relative to the sensitized group, and was close to the content value of the control group. It was demonstrated that GKK administration reduced the concentration of non-specific IgE antibodies in serum, thereby improving allergic symptoms.
The experimental results are shown in table 5 and the statistics are shown in fig. 7 for specific antibody IgE content in serum:
TABLE 5
The specific antibody IgE of sensitized mice (sensitized group) is obviously improved compared with the control group. The sensitized mice (high and low dose experimental groups) fed GKK2 by tube feeding had significantly lower levels of specific IgE antibodies in serum in vivo relative to the sensitized group (p < 0.05). It was demonstrated that GKK administration reduced the concentration of specific antibody IgE antibodies in serum, thereby improving allergic symptoms.
The experimental results are shown in fig. 8 for tissue sections. The sensitized mice (sensitized group) had significantly smaller airways compared to the control group. Tube fed GKK2 sensitized mice (high and low dose experimental groups) had insignificant tracheal constriction compared to the sensitized group and no significant differences compared to the control group. It was demonstrated that GKK administration can improve symptoms of bronchoconstriction, and has the effect of reducing the incidence of asthma.
The experimental results are shown in table 6 and the statistics are shown in fig. 9 for the glutathione reductase (GSH reduction) content:
TABLE 6
The glutathione reductase content of the sensitized mice group (asthma group) is greatly reduced, and the glutathione reductase content of the mice group sensitized after being fed with a higher dose of GKK2 is greatly improved, which is significantly higher than that of the asthma group (p < 0.05). It is shown that GKK administration can enhance immune function to improve allergic constitution, and has effect in reducing asthma attack.
In summary, the above experimental results prove that GKK2 can improve the problem of lumen shrinkage caused by tracheal contraction due to allergic asthma, reduce the content of non-specific and specific IgE antibodies in blood due to increased allergy, and increase the content of glutathione reductase in vivo. Showing that bifidobacterium lactis GKK2 has the efficacy of improving allergic asthma.
The invention provides a composition which comprises GKK and has the effect of relieving allergic asthma.
The composition further comprises an additive. In a preferred embodiment, the additive may be an excipient, preservative, diluent, filler, absorption enhancer, sweetener, or a combination thereof. The excipient may be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose, or a combination thereof. Preservatives may extend the shelf life of the pharmaceutical composition, for example benzyl alcohol, parahydroxybenzoic acid (parabens). The diluent may be selected from water, ethanol, propylene glycol, glycerin, or combinations thereof. The filler may be selected from lactose, milk sugar, high molecular weight polyethylene glycols or combinations thereof. The absorption enhancer is selected from dimethyl sulfoxide (DMSO), laurocapram, propylene glycol, glycerol, polyethylene glycol, or a combination thereof. The sweetener may be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame, or combinations thereof. In addition to the above-listed additives, other additives may be appropriately selected as required without affecting the pharmaceutical effect of the active substance of lactic acid bacteria.
The composition can be developed into different commodities in the field of medicine.
In a preferred embodiment, a composition is provided comprising bifidobacterium lactis GKK2 as a pharmaceutical product, feed, beverage, nutritional supplement, dairy product, food product or health food product.
The composition may take different forms depending on the needs of the subject. In a preferred embodiment, the composition is in the form of a powder, tablet, granulate, suppository, microcapsule, ampoul (amp), liquid spray or plug.
The compositions of the invention may be used in animals or humans. The composition comprising the active substance of lactic acid bacteria can be prepared in any pharmaceutical form without affecting the effect of the active substance of lactic acid bacteria, and administered to animals or humans in a suitable route depending on the pharmaceutical form.
Preparation of the composition
The GKK aspect of the invention, if applied to food applications, is exemplified by the following aspects of composition 1.
Composition 1: the lyophilized powder of GKK2 was taken as an active substance of lactic acid bacteria (20. 20 wt%), thoroughly mixed with benzyl alcohol (8 wt%) as a preservative and glycerin (7. 7 wt%) as a diluent, dissolved in pure water (65. 65 wt%), and stored at 4℃for use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.
GKK2 aspects of the invention the following aspects of composition 2 are illustrative examples if applied in liquid dosage form for medical use.
Composition 2: the lyophilized powder of GKK2 was taken as an active substance of lactic acid bacteria (20% wt%), and thoroughly mixed with benzyl alcohol (8% by weight) as a preservative, glycerin (7% wt%) as a diluent, sucrose (10% wt%) as a diluent, and dissolved in pure water (55% wt%) and stored at 4 ℃ for use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

Claims (18)

1. Lactobacillus bifidusBifidobacterium lactis) The bacterium is bifidobacterium lactis GKK2 with the preservation number of CGMCC No. 15205 and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms.
2. A composition comprising bifidobacterium lactis GKK as claimed in claim 1.
3. A composition comprising an active substance of bifidobacterium lactis GKK of claim 1;
wherein the active substance is prepared by the following method:
(a) Inoculating colonies of the bifidobacterium lactis GKK2 into a culture medium for solid-state culture to form bifidobacterium lactis thalli;
(b) Inoculating the bifidobacterium lactis cells cultured in the step (a) into a liquid culture medium for liquid culture;
(c) Centrifuging the liquid culture medium containing bifidobacterium lactis cells cultured in step (b) to obtain bacterial sludge; a kind of electronic device with high-pressure air-conditioning system
(d) And (3) freeze-drying the bacterial sludge obtained in the step (c).
4. A composition according to claim 3, wherein the culture conditions of step (b) are: the temperature is 35 to 50 ℃, the aeration is 0 to 1vvm nitrogen or carbon dioxide, the rotating speed is 10 to 100rpm, and the culture time is 16 to 24 hours.
5. A composition according to claim 3 wherein the pre-freeze temperature of the freeze drying of step (d) is from-196 to-40 ℃.
6. The composition of any one of claims 2 to 5 comprising an additive selected from the group consisting of: excipients, preservatives, diluents, fillers, absorption promoters, sweeteners or combinations thereof.
7. The composition of any one of claims 2 to 5, which is a pharmaceutical, feed or food product.
8. The composition of any one of claims 2 to 5, which is a beverage.
9. The composition of any one of claims 2 to 5, which is a nutritional supplement.
10. The composition of any one of claims 2 to 5, which is a dairy product.
11. The composition of any one of claims 2 to 5, which is a health food.
12. The composition of any one of claims 2 to 5 in the form of a powder, lozenge, granulate, suppository, microcapsule, ampoule, liquid spray or plug.
13. Use of bifidobacterium lactis GKK2 with a preservation number of CGMCC No. 15205 in the preparation of a pharmaceutical composition for improving allergic asthma.
14. The use of claim 13, wherein the improvement of allergic asthma is a decrease in respiratory resistance in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
15. The use of claim 13, wherein the improvement of allergic asthma is a decrease in the serum level of non-specific antibody IgE in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK.
16. The use of claim 13, wherein the improvement of allergic asthma is a decrease in the serum level of specific antibody IgE in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
17. The use of claim 13, wherein the improvement of allergic asthma is a reduction in the degree of bronchoconstriction in a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK2.
18. The use of claim 13, wherein the improvement of allergic asthma is an increase in the level of glutathione reductase in the liver tissue fluid of a subject administered bifidobacterium lactis GKK2 relative to a subject not administered bifidobacterium lactis GKK.
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